WO2021065665A1 - Method for producing three-dimensional cell structure - Google Patents

Abstract

The present invention addresses the problem of developing and providing a method for producing, in a simple and stable manner, a three-dimensional cell structure having an arbitrary three-dimensional shape and having high shape uniformity and physical strength.　A three-dimensional cell structure capable of solving said problem can be produced by placing a cell population that contains cell clusters having an average diameter of 50-750 µm inside a mold that has a wall surface having a liquid-permeable porous structure and by culturing the cell population therein.

Description

Manufacturing method of three-dimensional cell structure

The present invention relates to a method for producing a three-dimensional cell structure.

In recent years, there is an increasing possibility of practical application of a fundamental treatment method for intractable diseases and lifestyle-related diseases in regenerative medicine using stem cells having the ability to proliferate infinitely and differentiate into various cells. For example, in an in vitro experimental system, induction of differentiation from pluripotent stem cells into various cells such as nerve cells, myocardial cells, blood cells, and retinal cells has already been successful (Non-Patent Document 1). ).

On the other hand, there are still many issues to be solved for practical use regarding the regeneration of various organs and organs using stem cells. One of them is the problem in the construction of stem cell-derived three-dimensional structures. To use an artificial organ or artificial organ constructed by regenerative medicine technology as a transplant member to replace an organ or organ whose function has been lost due to malfunction or defect, etc., it imitates an organ or organ that has sufficient strength and substitutes. It is necessary to have a complicated three-dimensional shape.

In order to solve this problem, various methods for shaping cultured stem cells into a desired shape have been developed.

For example, Patent Document 1 discloses a method in which cell particles are put into a regulation frame portion to form a flat tissue and then press-molded. By applying an external stimulus called external pressure, a cell structure with high strength and biocompatibility can be formed. Further, a cell structure having a desired shape can be obtained by providing a groove portion, a through hole, a curved shape in a bent shape, or the like in the press molding portion.

Patent Document 2 discloses a method of molding a tubular structure using a silicon template using a cell structure containing a polymer (mosaic cell mass) as a raw material. There are four holes in the core holder of the lower base to improve the liquid diffusivity and improve the survival of cells.

Patent Document 3 discloses a method for obtaining a cell structure having a desired shape by culturing a cell mass after penetrating a cell mass through a ring-shaped sword ridge in order to form the cell into a tubular shape. ing.

Non-Patent Document 2 discloses a method of molding a single cell as a raw material using a mold made of agarose gel.

JP 2016-0000029 WO2016 / 068292 WO2013 / 073672

The method for producing a three-dimensional cell structure disclosed in the above prior art document has the following problems.

The mold used by the method described in Patent Document 1 is flat because it is pressed and molded, and does not have a mechanism for increasing the diffusivity of the culture solution. Therefore, there is a problem that a structure having an arbitrary shape cannot be easily manufactured. In addition, there is also a problem that the nutrient supply to the inside of the tissue tends to be insufficient because the diffusivity of the culture solution is low. In addition, applying external stimuli to cells by press molding carries the risk of adversely affecting tissue function.

Since the template used by the method described in Patent Document 2 has a tubular structure, a tubular structure can be produced as a three-dimensional cell structure. However, there is a problem that a structure having an arbitrary shape cannot be manufactured. In addition, there is also a problem that the nutrient supply to the inside of the tubular three-dimensional cell structure is still insufficient only by the drainage holes provided in the mold.

The method described in Patent Document 3 has a problem that the productivity is extremely low because it is necessary to individually fix the cell mass to the tubular sword ridge. Further, the shape of the three-dimensional cell structure is limited to a simple one, and there is also a problem that the cell density of the obtained three-dimensional cell structure is low and the shape tends to be non-uniform. Furthermore, since the cell mass is pierced and fixed with a sword mountain, there is a problem that the physical load on the cell is large and cell death is easily induced.

Since the method described in Non-Patent Document 2 uses a single cell as a raw material, the obtained three-dimensional cell structure tends to have a low cell density and a non-uniform shape, and there is a problem in the performance and yield of the cell structure.

As a result of diligent research conducted by the present inventors to solve the above problems, a cell mass having an average diameter in a specific range is placed inside a mold having a wall surface having a porous structure with liquid permeability and cultured. By doing so, we succeeded in easily and stably producing a cell structure having an arbitrary three-dimensional shape and having high shape uniformity and physical strength. The present invention has been completed based on these research results, and the following are provided.

(1) A method for producing a three-dimensional cell structure, wherein a cell population arranging step of arranging a cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less in the internal space of a template, and the cell population are described. The production method comprising a culturing step of culturing in a medium together with the mold, wherein the wall portion of the mold has a porous structure through which liquid can pass.
(2) The production method according to (1), wherein the cell population contains 50% or more of the cell mass.
(3) The production method according to (1) or (2), wherein the internal space of the mold is cylindrical.
(4) The production method according to any one of (1) to (3), wherein the mold has an inner wall portion inside the mold.
(5) The manufacturing method according to (4), wherein the hollow portion formed by the internal space and the inner wall portion of the mold is donut-shaped or tubular.
(6) Ratio of pore major axis (μm) and / or maximum diagonal line (μm) in the porous structure to the average diameter (μm) of the cell mass (average cell mass diameter / pore major axis and / or maximum diagonal line) The production method according to any one of (1) to (5), wherein is greater than 1.0 and less than or equal to 3.0.
(7) The production method according to any one of (1) to (6), wherein the pore opening ratio in the porous structure is 40% or more and 90% or less.
(8) The method according to any one of (1) to (7), wherein in the cell population placement step, the cell population containing the cell mass is filled to 60% or more and 95% or less of the volume of the mold internal space. Production method.
(9) The production method according to any one of (1) to (8), wherein in the cell population placement step, the cell mass is laminated without applying artificial pressure to the template.
(10) The production method according to any one of (1) to (9), wherein the culture period in the culture step is 4 hours or more and less than 28 days.
(11) The production method according to any one of (1) to (10), further comprising a separation step of separating the three-dimensional cell structure obtained after the culture step and the template.
(12) The production method according to (11), wherein the cell contact surface of the template is made of a cell non-adhesive material.
(13) The method according to any one of (1) to (12), further comprising a cell population preparation step of preparing a cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less prior to the cell population placement step. Manufacturing method.
(14) The cell according to any one of (1) to (13), wherein at least one cell constituting the cell mass is selected from the group consisting of mesenchymal stem cells, fibroblasts, endothelial cells and chondrocytes. Manufacturing method.
(15) The production method according to (14), wherein the mesenchymal stem cells are derived from bone marrow, fat, fetal appendages or dental pulp.
(16) A cell population which is a three-dimensional cell structure and contains a cell mass having an average diameter of 50 μm or more and 750 μm or less is placed in the inner space of a template, and then the cell population is cultured in a medium together with the template. The three-dimensional cell structure obtained by the above method, wherein the wall portion of the template has a porous structure through which liquid can pass.
This specification includes the disclosure of Japanese Patent Application No. 2019-178749, which is the basis of the priority of the present application.

According to the method for producing a three-dimensional cell structure of the present invention, a highly uniform cell structure having an arbitrary three-dimensional shape can be easily produced.
According to the method for producing a three-dimensional cell structure of the present invention, a three-dimensional cell structure having high shape uniformity and high physical strength can be stably produced.

It is a figure which shows the manufacturing flow of the 3D cell structure of this invention. It is a figure which shows an example of the template used in the 3D cell structure manufacturing method of this invention. It is a figure which showed the result of observing the state of the cell culture in the culture step after the cell population arrangement step of Example 1. FIG. A is a diagram immediately after the cell population placement step, and B is a diagram on the 7th day of culturing. In the figure, the arrows indicate the arranged cell population (mainly the cell mass). It is a figure which showed the result of observing the state of the cell culture in the culture step after the cell population arrangement step of Example 2. FIG. A is a diagram immediately after the cell population placement step, and B is a diagram on the 7th day of culturing. In the figure, the arrows indicate the arranged cell population (mainly the cell mass). It is a figure which showed the result of observing the state of the cell culture in the culture step after the cell population arrangement step of the comparative example 1. FIG. A is a diagram immediately after the cell population placement step, and B is a diagram on the 7th day of culturing. In the figure, the arrows indicate the arranged cell population (mainly the cell mass). It is a figure which showed the result of having confirmed the state of the structure (arrow) at the time of removing the mold of Example 2. In the figure, the arrowhead indicates a stainless steel cylinder installed as an inner wall in the space inside the mold. It is a figure which showed the state of the three-dimensional cell structure after removal of the template and the inner wall part of Example 2. FIG. It is a figure which showed the state of the tissue (arrow) at the time of removing the mold of the comparative example 1. FIG. In the figure, the arrowhead is a stainless steel cylinder installed as an inner wall in the space inside the mold.

1. 1. Method for producing three-dimensional cell structure 1-1. Outline The first aspect of the present invention is a method for producing a three-dimensional cell structure. In the production method of the present invention, a cell population containing a cell mass having an average diameter in a specific range is placed inside a mold having a wall surface having a porous structure with fluid permeability. As a result, cell clusters can be arranged at high density in the internal space. Further, it is sufficient to fill and stack the cell population in the space inside the template, and therefore there is an advantage that there is no physical load such as external pressure on the cells. According to the production method of the present invention, a cell structure having a high cell density having an arbitrary three-dimensional shape can be easily produced. As a result, it becomes possible to stably produce a three-dimensional cell structure having high strength and uniformity, improve the yield, and enable mass production of the three-dimensional cell structure.

1-2. Definition of terms and their structure The following terms frequently used in the present specification are defined, and their structures are specifically described. Unless otherwise specified, the following definitions described in this section are common to other aspects of the present invention.

(1) Three-dimensional cell structure As used herein, the term "three-dimensional cell structure" refers to a three-dimensional structure composed of a cell population. Therefore, it is composed of a cell layer such as a cell sheet and does not include a planar structure having a substantially uniform thickness. The three-dimensional cell structure may be composed only of cells, but may contain substances other than cells. Examples of substances other than cells include, but are not limited to, extracellular matrix, a part of a template, and the like. Further, the three-dimensional cell structure may have a scaffold or may be scaffold-free. It is preferably scaffold-free. As used herein, "scaffold" means a scaffold for growing cells. The scaffold is preferably composed of a material that is absorbed by a living body, and examples of such a material include, but are not limited to, polylactic acid, collagen, and the like. Each cell constituting the three-dimensional cell structure may be of the same or different types.

The shape of the three-dimensional cell structure may be either a fixed shape or an indefinite shape. Examples of the standard shape include a spherical shape, an elliptical shape, a cubic shape, a rectangular shape, a polyhedral shape, a conical shape, a pyramidal shape, a cylindrical shape, a prismatic shape, a spiral shape, a donut shape shape, and a tubular shape. Not limited to.

(2) Cell The “cell” in the present specification is a constituent unit of a three-dimensional cell structure, and means an adhesive cell capable of cell-cell adhesion or cell-extracellular matrix adhesion unless otherwise specified. .. Adhesion of adherent cells to the cell culture substrate, cells, or extracellular matrix is referred to as "cell adhesion".

The type of cell in the present specification is not limited. For example, cells constituting a living tissue, cells derived from cells constituting a living tissue, stem cells, or cells differentiated from stem cells can be mentioned. Moreover, the cell in this specification may be a cell isolated from a living tissue.

In the present specification, the "living tissue" refers to various tissues constituting the living body of an organism. For example, epithelial tissue, endothelial tissue, connective tissue, muscle tissue, nerve tissue and the like can be mentioned. Examples of cells constituting these tissues include epithelial cells, endothelial cells, fibroblasts, muscle cells, nerve cells, and chondrocytes.

"Stem cell" refers to a cell that has the ability to differentiate into various cells and the ability to self-renew. For example, adult stem cells, pluripotent stem cells and the like can be mentioned.

An "adult stem cell" is a stem cell that exists in each adult tissue, has not completed final differentiation, and has a certain degree of pluripotency, and is a somatic stem cell or tissue. It is also called a sex stem cell. Examples thereof include neural stem cells, intestinal epithelial stem cells, hematopoietic stem cells, hair follicle stem cells, pigment stem cells, mesenchymal stem cells and the like. "Neural stem cells" are mainly nerve cells and cells having the ability to differentiate into glial cells. "Intestinal epithelial stem cells" are cells having the ability to differentiate into epithelial cells that mainly constitute the inner wall of the digestive tract such as the small intestine and the large intestine. "Hematopoietic stem cells" are cells having the ability to differentiate into blood cells such as erythrocytes, leukocytes, and platelets.

"Hair follicle stem cells" are cells having the ability to differentiate into hair follicle epithelial cells such as hair follicle stem cells and hair root sheath cells, as well as fat gland cells and basal cells. Further, the "chromophore stem cell" is a cell having an ability to differentiate into a chromatophore mainly. As the adult stem cells used in the present specification, commercially available cells or cells that have been distributed may be used, or newly prepared cells may be used.

"Mesenchymal stem cells (MSCs)" show adhesion to plastic under culture conditions in standard medium, and are positive for surface antigens CD73 (5-Nucleotidase) and CD90 (Thy-1). , And CD45 (PTPRC (Protein tyrosine phosphatase, receptor type, C)) is a negative cell. Mesenchymal stem cells may have the ability to differentiate into cells belonging to the mesenchymal system such as osteoblasts, adipocytes, muscle cells, and chondrocytes. Mesenchymal stem cells can be collected from, for example, bone marrow, fat, fetal appendages, pulp and the like. The "fetal appendage" is an organ necessary for the foetation to develop in the uterus, and examples thereof include placenta, umbilical cord, amniotic membrane, egg membrane, chorion, decidua, and amniotic fluid. The mesenchymal stem cells used herein are mesenchymal stem cells that have been genetically modified or have not been genetically modified, and are preferably non-genetically modified mesenchymal stem cells.

The "ratio of cells positive for surface antigen" in the present specification indicates the ratio of cells positive for surface antigen analyzed by flow cytometry. In the present specification, "the ratio of cells in which the surface antigen is positive" may be described as "positive rate". Further, the “ratio of cells in which the surface antigen is negative” in the present specification indicates the ratio of cells in which the surface antigen is negative in the surface antigen analyzed by flow cytometry. In the present specification, "the ratio of cells in which the surface antigen is negative" may be described as "negative rate". The ratio of cells negative to each surface antigen (negative rate) can be calculated by the formula "negative rate (%) = 100-positive rate".

A "pluripotent stem cell" is a pluripotent (pluripotent) cell capable of differentiating into all types of cells constituting a living body, and is cultured in vitro under appropriate conditions. A cell that can continue to grow indefinitely while maintaining pluripotency. For example, embryonic stem cells (ES cells: embryonic stem cells), embryonic germ stem cells (EG cells: embryonic stem cells), germline stem cells (GS cells: Germline stem cells), and induced pluripotent stem cells (iPS cells: induced). Pluripotent stem cells) and the like. "ES cells" are pluripotent stem cells prepared from early embryos. "EG cells" are pluripotent stem cells prepared from fetal primordial germ cells (Shamblott MJ et al., 1998, Proc. Natl. Acad. Sci. USA., 95: 13726-13731. ). "GS cells" are pluripotent stem cells prepared from the cell testis (Conrad S., 2008, Nature, 456: 344-349). The "iPS cell" refers to a pluripotent stem cell that can be reprogrammed to bring the somatic cell into an undifferentiated state by introducing a gene encoding a small number of reprogramming factors into the differentiated somatic cell. ..

As the pluripotent stem cell used in the present specification, a commercially available cell or a cell for sale may be used, or a newly prepared cell may be used. Although not limited, iPS cells or ES cells are preferable as the pluripotent stem cells when used in each invention of the present specification.

When the iPS cells used in the present specification are commercially available products, for example, 253G1 strain, 201B6 strain, 201B7 strain, 409B2 strain, 454E2 strain, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS -RIKEN-12A strain, Nippons-B2 strain, TkDN4-M strain, TkDA3-1 strain, TkDA3-2 strain, TkDA3-4 strain, TkDA3-5 strain, TkDA3-9 strain, TkDA3-20 strain, hiPSC38-2 A strain, MSC-iPSC1 strain, BJ-iPSC1 strain and the like can be used. In addition, newly prepared clinical grade iPS cells may be used.

When the iPS cells used in the present specification are newly prepared cells, the combination of genes of the reprogramming factors to be introduced is not limited, but is, for example, OCT3 / 4 gene, KLF4 gene, SOX2 gene and c. -Myc gene combination (YuJ, et al. 2007, Science, 318: 1917-20.), OCT3 / 4 gene, SOX2 gene, LIN28 gene and Nanog gene combination (Takahashi K, et al. 2007, Cell, 131: 861-72.) Can be used. The form of introduction of these factors into cells is not particularly limited, and examples thereof include gene transfer using a plasmid, introduction of synthetic RNA, and direct introduction as a protein. Further, iPS cells prepared by a method using microRNA, RNA, low molecular weight compounds and the like may be used. In addition, newly prepared clinical grade iPS cells may be used.

When the ES cells used in the present specification are commercially available products, for example, KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SEES1 strain, and SEES2 strain are not limited. , SEES3 strain, SES-4 strain, SES-5 strain, SES-6 strain, SES-7 strain, HUES8 strain, CyT49 strain, H1 strain, H9 strain, HS-181 strain and the like can be used.

The cell-derived species in the present specification may be a multicellular organism. It is preferably an animal, more preferably a mammal. For example, rodents such as mice, rats, hamsters, guinea pigs, gerbils, livestock or pets such as dogs, cats, rabbits, cows, horses, pigs, sheep, goats, ferrets, and humans, crab monkeys, red-tailed monkeys, common marmosets. , Japanese monkeys, gorillas, chimpanzees and other primates. Especially preferably human.

The cells herein may be autologous, allogeneic or heterologous cells, preferably allogeneic cells. The cells herein may be cultured cells or passaged cells.

(3) Cell mass In the present specification, the “cell mass” is a mass-like aggregate formed by cell aggregation. Also called cell agglutinin or spheroid. "Cell aggregation" means that a plurality of cells are three-dimensionally aggregated to form an agglutination. There are agglutination by different cells and agglutination by the same cell, whichever is used herein. Aggregation by the same cell includes the case where agglomerates are formed by the proliferation of one cell. The mechanism of cell aggregation includes, but is not limited to, adhesion between cells via membrane proteins, cell membranes, extracellular matrix, and adhesion between cells via cadherin on the cell surface.

The shape of the cell mass is not particularly limited. Since the cell mass formed by cell aggregation usually has a substantially spherical shape, a substantially spherical shape is preferable.

The size of the cell mass is 50 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, 220 μm or more, 250 μm or more, 300 μm or more, 350 μm or more, or 400 μm or more, and 750 μm or less, 700 μm or less, 650 μm or less, 600 μm. Hereinafter, it is 550 μm or less, 500 μm or less, or 450 μm or less. Here, the “diameter” in the present specification means the diameter (μm) of a substantially spherical cell mass, and the “average diameter” in the present specification is an arithmetic mean (μm) of the diameters of a plurality of cell clusters. ) Means. The measurement of the average diameter of the cell mass can be calculated from an image obtained by photographing the cell mass using, for example, a microscope and imaging software (for example, cellSense manufactured by OLYMPUS). Specifically, the average diameter of the cell mass can be obtained by measuring the diameter from any three directions of the photographed image of the cell mass and calculating the average value thereof. When the average diameter of the cell mass is within the above range, it is possible to reduce and homogenize the gap between the contact portions between the cell masses that may occur when the cell mass is placed in the inner space of the template, and as a result. , It is possible to prevent the occurrence of cracks in the cell culture in the culture step. Therefore, the cell density and shape uniformity of the obtained three-dimensional cell structure can be improved. If the average diameter of the cell mass is less than 50 μm, the shape of the obtained three-dimensional cell structure becomes non-uniform, which is not preferable. Further, when the average diameter of the cell mass is larger than 750 μm, the gap between the contact portions between the cell masses in the inner space of the template becomes large, so that the cell masses are unevenly fused in the culture step, resulting in cracks and a rough surface. It is not preferable because a cell culture having the above is formed.

(4) Cell population As used herein, the term "cell population" refers to a population consisting of a plurality of cells including at least the cell mass. The cell population may consist solely of cell clusters or may include a single cell or several cells. Each cell mass and / or each cell constituting the cell population can exist separately from each other in a liquid such as a culture solution without cell adhesion in the cell population. The liquid containing the cell population in this case is often referred to herein as a "cell suspension."

Among all cells and cell clusters contained in the cell population, the ratio of cell clusters within the above size range is 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more. , 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. When the cell population contains 50% or more of the cell mass, the homogeneity of the cell population is enhanced, and as a result, the arrangement of the cell mass in the inner space of the template becomes more uniform, and the cell mass fuses more uniformly in the culture step. Therefore, it is possible to obtain a three-dimensional cell structure having no visible cracks and further improved shape uniformity.

The cell population of the present invention can contain any number of cell clusters of 2 or more, eg, 1.0 × 10 ¹ , 1.0 × 10 ² , 2.0 × 10 ² , 5, .0 × 10 ² pcs, 1.0 × 10 ³ pcs, 2.0 × 10 ³ pcs, 5.0 × 10 ³ pcs, 1.0 × 10 ⁴ pcs, 2.0 × 10 ⁴ pcs, 5.0 × 10 ⁴ pieces, 1.0 × 10 ⁵ pieces, 1.0 × 10 ⁶ pieces, 1.0 × 10 ⁷ pieces, 1.0 × 10 ⁸ pieces, 1.0 × 10 ⁹ pieces, 1.0 × 10 It can include, but is not limited to, ^{10 or more or less cell clusters.}

The plurality of cell clusters or the plurality of cells contained in the cell population may be derived from the same species or different species from each other.

The cell suspension used in the present specification may contain any component other than cells, and examples thereof include salts, sugars, proteins, amino acids, medium components, buffers, and the like. Not limited. The pH of the cell suspension can be near neutral, for example, pH 5.5 or higher, pH 6.0 or higher, pH 6.5 or higher or pH 7.0 or higher, and pH 10.5 or lower, pH 9 or higher. It can be 5.5 or less, pH 8.5 or less, or pH 8.0 or less, but is not limited thereto.

(5) Medium As used herein, the term "medium" refers to a liquid, semi-solid or solid substance prepared for culturing cells. As a general rule, it contains more than the minimum necessary components essential for cell proliferation and / or maintenance. Unless otherwise specified, the medium used in the present specification corresponds to a liquid medium for animal cells used for culturing animal-derived cells. In addition, the medium is not limited, but is BME medium, BGJb medium, CMRL1066 medium, Glasgo MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscover's Modified Dulvecco'S Medium), Medium Medium 199 medium. αMEM medium, DMEM medium (Dulvecco'S Modified Eagle'S Medium), ham F10 medium, ham F12 medium, RPMI 1640 medium, Fisher'S medium, and a mixed medium thereof (for example, DMEM / F12 medium (Dulvecco'S Medium)). Eagle'S Medium / Nutrition Mixture F-12 Ham)) and the like can be used. As the DMEM / F12 medium, the weight ratio of the DMEM medium and the ham F12 medium is preferably in the range of 60/40 or more and 40/60 or less, for example, 58/42, 55/45, 52/48, 50/50, 48 /. A medium mixed with 52, 45/55, 42/58, etc. is used. In addition, the medium used for culturing human iPS cells and human ES cells can also be preferably used.

The medium used in the present invention may be a medium containing serum or a serum-free medium. Serum is, but is not limited to, fetal bovine serum (FBS), horse serum and the like. In the medium containing serum, the final concentration of serum in the medium is 1% or more, 2% or more, 3% or more, 4% or more or 5% or more, and 20% or less, 18% or less, 16% or less. , 14% or less, 12% or less or 10% or less, more preferably 9% or less, still more preferably 8% or less. The serum-free medium is not limited, but is, for example, STK1 or STK2 (manufactured by DS Pharma Biomedical), EXPREP MSC Medium (manufactured by Biomimetic Sympathies), Corning stemgro human adhesive stem cell medium (manufactured by Corning), etc. It may be a commercially available serum-free medium.

The medium used in the present specification can be prepared as a medium specific to various cells by adding various culture additives. A "culture additive" is a substance added to a medium for the purpose of culturing. Specific examples of culture additives include, but are not limited to, serum, serum substitute reagents, insulin, transferrin, selenium, cytokines, growth factors, albumin, sodium hydrogen carbonate, fatty acids, amino acids (eg, non-essential amino acids), L-ascorbin. Examples include acids, vitamins, antioxidants, 2-mercaptoethanol, pyruvate, buffers, inorganic salts, polysaccharides, antibiotics and the like. Insulin, transferrin, and cytokines may be of natural origin isolated from animal (preferably human, mouse, rat, bovine, horse, goat, etc.) tissues or sera, or genetically engineered. It may be a recombinant protein. The growth factors are not limited, but are, for example, FGF2 (Basic fiberblast growth factor-2), EGF (Epidermal growth factor), TGF-β1 (Transforming growth factor-β1), VEGF (VEGF). (Platelet-developed growth factor), Activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF and IGFBP-7 can be used. Antibiotics include, but are not limited to, penicillin, streptomycin, amphotericin B and the like. A particularly preferred growth factor as a culture additive for the medium used in the present invention is FGF2. One or more culture additives can be contained in the medium.

Culture additives can be added to the medium in the form of solutions, derivatives, salts, mixed reagents and the like. For example, L-ascorbic acid may be added to the medium in the form of a derivative such as magnesium 2-ascorbic acid, and selenium may be added to the medium in the form of a selenium salt (sodium selenite, etc.). May be good. Insulin, transferrin, and selenium can also be added to the medium in the form of an ITS reagent (insulin-transferrin-selenium). It is also possible to use a commercially available medium to which at least one selected from L-ascorbic acid, insulin, transferrin, selenium and sodium hydrogen carbonate has already been added. Commercially available media containing insulin and transferase include CHO-S-SFM II (Life Technologies Japan Co., Ltd.), Hybridoma-SFM (Life Technologies Japan Co., Ltd.), eRDF Dry Powered Media (Life Technologies Japan Co., Ltd.), and UltraCULTURE. ^TM (BioWittaker), UltraDOMA ^TM (BioWhittaker), UltraCHO ^TM (BioWhittaker), UltraMDCK ^TM (BioWhiteker), STEMPRO (Registered Trademarks) (Veritas) and the like.

(6) Mold As used herein, the term "template" refers to a molding mold for producing a three-dimensional cell structure. Due to its nature, the template has an internal space (cavity: sometimes referred to as "internal space" in the present specification), and the internal space is usually the appearance of a three-dimensional cell structure. It has a shape that exhibits.

The outer shape of the mold is not particularly limited. It may have a shape similar to or dissimilar to the three-dimensional cell structure to be produced. On the other hand, the shape of the internal space has a shape similar to the external shape of the three-dimensional cell structure to be manufactured, in whole or in part. Therefore, the shape of the internal space can be made into a desired shape according to the external shape of the three-dimensional cell structure. For example, if it is desired to produce a cylindrical three-dimensional cell structure, the internal space of the template may be cylindrical. Generally, when the wall portion of the mold is thin, the outer shape and the shape of the inner space are similar. Therefore, it approaches the similar shape of the three-dimensional cell structure that also manufactures the outer shape.

The mold can have an inner wall portion (core) arranged in the internal space. In this case, in the template, the internal space corresponds to a concave portion (female type) and the inner wall portion corresponds to a convex portion (male type), and the hollow portion formed between the internal space and the inner wall portion is the target three-dimensional cell. It exhibits the shape of a structure. The shape of the inner wall portion can be a desired shape according to the outer or inner shape of the three-dimensional cell structure. For example, when it is desired to produce a donut-shaped or tubular three-dimensional cell structure, the internal space of the template may be cylindrical and the inner wall may be cylindrical, which is smaller than the internal space. A donut-shaped or tubular three-dimensional cell structure can be produced by adjusting the amount of the cell population to be filled in the donut-shaped or tubular cavity formed between the internal space and the inner wall portion.

The material of the mold may be any material as long as it can have a porous structure through which all or part of the wall can pass, and is not particularly limited. Since the template is immersed in the medium as described later, it is desirable that the template is a water-insoluble material or a non-soluble material. "Water-insoluble material" means a material that is insoluble in water or an aqueous solution. Includes inanimate systems, biological systems, and mixtures thereof. An inanimate water-insoluble material is a water-insoluble material whose material is not directly derived from a living organism. For example, plastic (including chemical fibers), glass, metal, silicon, synthetic rubber, ceramics and the like can be mentioned. For plastics, for example, polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), polyurethane (PU), polyvinylidene chloride (PVC), polyvinylidene chloride (PVDC), polycarbonate (PC). ), Polysulfone (PSU), polyarylate (PAR), polyamide (nylon), polyvinyl alcohol (PVA) and the like can be used. If it is a metal, for example, a pure metal such as gold (Au), platinum (Pt), silver (Ag), copper (Cu), nickel (Ni), titanium (Ti), aluminum (Al), tartar (Ta), etc. Other examples include alloys such as stainless steel (SUS), cobalt alloys, and nitinol (NiTi). Biological water-insoluble materials refer to water-insoluble materials produced directly by living organisms. Examples thereof include natural fibers (cellulose, keratin, fibroin, etc.), natural resins (eg, natural rubber or lacquer), bones (including coral skeleton, spongy skeleton, etc.), teeth, horns, xylem, charcoal, and the like. The "non-soluble material" means a material that does not dissolve in water or an aqueous solution under normal temperature and pressure. Although not limited, it is usually of biological origin. Examples include polysaccharide macromolecules (eg, agar, mannan), gelled proteins (eg, gelatin, collagen), or mixtures thereof.

The mold may be a composite material composed of two or more different materials. For example, in the wall portion, a material facing the outside of the mold and a material facing the internal space may be different materials. Such a structure is convenient because when each surface constituting the wall portion is arranged in a different environment, an appropriate material can be used according to the environment. For example, the wall surface of the interior space is a cell contact surface that comes into direct contact with cells. In this case, the inner space wall surface is preferably made of a non-cell adhesive material. This is because when the cell contact surface is a material having high cell adhesion, it becomes difficult to separate the three-dimensional cell structure and the template after production. Examples of the cell non-adhesive material include stainless steel, aluminum, polypropylene and the like. Further, as the internal space wall surface, a material having a surface coated with a substance that inhibits cell adhesion can be adopted. Examples of the substance that inhibits cell adhesion include, but are not limited to, agar, poly (2-hydroxyethyl methacrylate), polyethylene glycol, and the like.

As a feature of the mold used in the present invention, it has a porous structure that allows liquid to pass through all or part of the wall portion as described above. "Wall portion" refers to a portion of the mold that constitutes the wall surface (including the side surface, bottom surface, and top surface) of the internal space. Further, "liquid passable" means that the liquid can pass through the wall portion. Due to this liquid permeability in the wall part, the liquid can freely move between the outside and the inside space of the part. The "liquid" referred to here is mainly water or an aqueous solution, although it is not limited. Aqueous solution includes medium and buffer. As the buffer, a darbecolinic acid buffer (DPBS), an Earl balanced salt solution (EBSS), a Hanks balanced salt solution (HBSS), a phosphate buffer (PBS) and the like can be used, but the buffer is not limited thereto.

In the present specification, the "porous structure" means a structure having a plurality of pores. All or part of the holes are open on the surface of the material having a porous structure. Examples of the porous structure include a mesh structure and a structure in which a bubble structure has a perforated surface. The shape of the pores in the porous structure is not limited. It may be circular, substantially circular, elliptical, polygonal, substantially polygonal, amorphous, or a combination thereof. It is preferable that the upper limit of the individual pore size is a size that the cell mass cannot pass through, and that the cell population can be supported when the cell population is laminated in the template internal space and the template can be self-supporting. Further, the lower limit is preferably a size capable of maintaining liquid permeability without causing clogging of the holes. For example, when the average diameter of the cell mass is "d" and the major axis and / or the maximum diagonal of the pore is "D", the ratio of the major axis and / or the maximum diagonal of the pore to the average diameter of the cell mass (d /). D) is greater than 1.0, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, or 2.0 or more, and 3.0 or less, 2. It may be 9 or less, 2.8 or less, 2.9 or less, 2.6 or less, or 2.5 or less. If the ratio (d / D) is 1.0 or less, the cell mass arranged in the mold internal space may pass through the pores, which is not preferable. Further, if the ratio (d / D) is larger than 3.0, when the cell mass is arranged in the template internal space, the pores may be clogged by the cell mass, and sufficient liquid permeability may not be ensured. , Not preferable. As an example of a specific hole size, the major axis and / or the maximum diagonal of the hole is 50 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, 220 μm or more, 250 μm or more, or 300 μm or more, and 750 μm or less, 700 μm or less, 650 μm or less, It is 600 μm or less, 550 μm or less, 500 μm or less, or 450 μm or less.

The pore opening ratio in the porous structure region of the mold wall portion means the ratio of the pore portion to the total area. For example, in the case of a mesh structure or the like, when the average diameter of the hole portion is “A” and the wire diameter is “a”, ^{it becomes (A / A + a) 2} × 100 (%). The aperture ratio in the present specification is 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more, and 90% or less, 85% or less, It is preferably 80% or less, or 75% or less. By setting the aperture ratio to 40% or more and 90% or less, it is possible to secure the nutrient supply and support for the cell population in the internal space, and to form a three-dimensional cell structure while maintaining the viable state. If the aperture ratio is less than 40%, sufficient fluid permeability to the internal space cannot be maintained during static culture, and the nutrient supply to the cell population in the internal space cannot be secured, which is not preferable. Further, if the aperture ratio is larger than 90%, the template strength for supporting the cell population in the internal space may be insufficient, which is not preferable.

The material and characteristics of the inner wall portion described above may be the same as those of the mold, or may be completely or partially different. For example, the cell adhesion surface of the inner wall portion is preferably a cell non-adhesion material as in the template. On the other hand, the inner wall portion may not have liquid permeability, or may have a porous structure having liquid permeability similar to the mold. When it has liquid permeability, the specific porous structure may be the same as or different from that of the mold. For example, there is a case where the side surface of the mold wall portion has a mesh structure and the inner wall portion has a porous structure having a bubble structure.

The template having the above structure (including the inner wall portion) can also be used as a template capable of embodying the method for producing a three-dimensional cell structure of the present invention, that is, a template for producing a three-dimensional cell structure.

1-3. Method The flow of the method for producing a three-dimensional cell structure of the present invention is shown in FIG. The production method of the present invention includes a cell population arrangement step (S0102) and a culture step (S0103) as essential steps, and a cell population preparation step (S0101) and a separation step (S0104) as selection steps. Hereinafter, each step will be specifically described.

(1) Cell Population Preparation Step The “cell population preparation step” (S0101) is a step of preparing a cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less. This step is a selective step carried out prior to the cell population placement step.

The method for preparing the cell population is not particularly limited. Basically, it may be prepared so that a cell mass having a predetermined size is contained in the cell suspension based on a method for preparing a cell suspension known in the art. As a method for preparing a cell suspension containing a cell mass, for example, a cell obtained by an adhesive culture method or a tissue piece collected from a living body or the like is used, for example, a Prime Surface (registered trademark) plate (manufactured by Sumitomo Bakelite Co., Ltd.) or the like. There is a method of culturing on a cell non-contact substrate. At that time, by seeding under the condition that the cells keep in contact with each other on the surface of the base material, the cells can spontaneously aggregate and form a cell mass. Other methods of preparing cell suspensions containing cell clusters include hanging drop methods, chemical treatments and / or methods in which the droplets of the cell suspension are maintained in a suspended form and the cells aggregate in the droplets. There are a method of preparing by physical treatment, a method of preparing by suspension culture method, and the like.

In the present specification, the "adhesive culture method" refers to a culture method in which cells are adhered to a culture vessel or the like and, in principle, are grown in a single layer. The "chemical treatment" refers to chemically treating monolayer cells and tissue pieces obtained by the adhesion culture method with a cell release agent used for detachment of adherent cells and cell dispersion of tissues. As the cell exfoliating agent, trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA) and the like can be used, but the cell exfoliating agent is not particularly limited. As the cell exfoliating agent, a commercially available cell exfoliating agent may be used. For example, trypsin-EDTA solution (manufactured by Thermo Fisher Scientific), TrypLE Select (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Stemcell Technology), etc.

In addition, "physical treatment" promotes the separation of cell adhesion by applying a mechanical external force such as peeling treatment or suspension treatment to monophasic cells or tissue pieces whose cell adhesion is partially separated by chemical treatment or the like. The process of causing. In the method for preparing a cell population by chemical treatment and / or physical treatment, the amount of enzyme and reaction when dispersing cells so that a part of the cells remains in the cell suspension as a cell mass having the average diameter. It must be prepared in consideration of temperature, reaction time, etc., additional strength of external force, number of additions, etc. as appropriate.

Further, in the present specification, the "suspension culture method" is one of the cell culture methods, and refers to a method in which cells are grown in a floating state in a medium. In this method, the cultured cells exist as a cluster of cells aggregated in the culture medium. Therefore, when preparing a cell mass of a predetermined size, the suspension culture method is particularly preferable. In the suspension culture method, cell aggregation is promoted by administering a drug that inhibits or suppresses the activity of a signal transduction factor involved in the intracellular myosin signal transduction pathway into the medium, and conversely, the activity of the same factor is promoted or Cell aggregation can be suppressed by administering the amplifying agent in the medium. By utilizing these actions, it is possible to control the promotion and suppression of aggregation of suspended cultured cells, and it is also possible to produce cell clusters having an average diameter of 50 μm or more and 750 μm or less. Examples of specific methods for preparing a cell mass of a desired size by the suspension culture method include, but are not limited to, for example, International Publication No. WO2016 / 121737, Japanese Patent Application Laid-Open No. 2019-118279, International Publication No. WO2019 / 131941 and. The method of disclosure can be referred to in International Publication No. WO 2019/131942. After the preparation, the cell population may be washed with a buffer or a medium and then suspended in them to obtain a cell suspension, or the prepared culture solution may be used as it is as a cell suspension.

(2) Cell Population Arrangement Step The “cell population arrangement step” (S0102) is a step of arranging a cell population including a cell mass in the internal space of the template.

In this step, "arrangement" means filling the cell population including the cell mass into the inner space of the template or the cavity formed when the inner wall portion is arranged in the inner space. The cell population before being placed in the template exists as a cell mass or a cell suspension in which cells are dispersed in a solution. However, by arranging the cells inside the template in this step, the cell clusters and cells contained in the cell population gather, and when they come into contact with each other, they are formed into a desired three-dimensional cell structure.

The placement method in the mold internal space is not limited. For example, the cell suspension may slowly flow into the template internal space. The inflow of the cell suspension can also be carried out with the template immersed in the medium. As the cell suspension, in addition to the cell suspension prepared in the cell population preparation step, a commercially available cell suspension containing a cell mass having a predetermined size can also be used.

The amount of cell population filled in the mold internal space is not particularly limited. It may be appropriately determined in consideration of the mold internal space, the shape and size of the target three-dimensional cell structure, and the like. As used herein, the term "filling" means a state in which a cell population is placed inside a template, then allowed to stand, and the cells become close-packed by natural sedimentation. In the case of this step, for example, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 85% or more, 90% or less, 91% or less, 92% or less of the volume of the mold internal space, It may be filled up to 93% or less, 94% or less, or 95% or less. By filling the mold so that it is 60% or more and 95% or less of the volume of the internal space of the mold, the yield when producing a three-dimensional cell structure can be further improved. On the other hand, when the filling rate is less than 60% of the volume of the inner space of the mold, the gap between the contact portions between the cell masses becomes large due to the presence of the cell masses at a low density, and cracks and rough surfaces are formed in the culture step. It is not preferable because it may form a cell culture having the cells. Further, when the filling rate is larger than 95% of the volume of the mold internal space, the physical load due to the contact between the cell clusters increases due to the presence of the cell clusters at an ultra-high density, which is not preferable.

The arrangement of the cell population in the template can be performed by one or multiple operations. In the case of a plurality of times, the second placement is performed after a predetermined time has elapsed after the first placement step. The amount of cell population arranged in each round may be the same or different. Furthermore, the cell masses and cell types that make up the cell population, or the average diameter of the cell masses may be the same or different each time.

In the arrangement in the mold internal space, the cell mass or the cell mass is arranged by a method of spontaneously dropping the cell mass or cells, a method of applying artificial pressure from the cell population inlet, or a method of suction arrangement from the outside of the template and / or the inside of the inner wall. Stacking cells can be mentioned, but is not limited to these methods. However, if an external force is applied to the cells during placement, damage to the cells may occur, so placement by free fall without applying artificial pressure or suction force is preferable. Further, in the present specification, "stacking" means arranging a plurality of the cell masses and cells in the internal space of the template so as to maintain a state of being in contact with each other in the direction of gravity.

(3) Culturing Step The “culturing step” (S0103) is a step of culturing a cell population arranged in the template internal space after the cell population arranging step in a medium together with the template. "With a template" means that a cell population arranged in a template internal space is immersed in a medium together with the template and cultured. Since the mold wall portion has a porous structure with liquid permeability, the cell population in the mold internal space can be cultured by the inflow and outflow of the medium through the pores thereof. Within the interior space, the cell masses and / or cells adhere to each other via contact and integrate within the interior space of the mold. As a result, the desired three-dimensional cell structure is formed inside the template after this step.
The shape and size of the culture tank containing the medium are not limited as long as the mold can be immersed in the tank.

The culture conditions such as the culture temperature, time, and CO _{2 concentration in this step are not particularly limited.} It may be done within the scope of the conventional law in the field. For example, the lower limit of the culture temperature may be 20 ° C. or higher or 35 ° C. or higher, and the upper limit may be 45 ° C. or lower or 40 ° C. or lower, but is preferably 37 ° C. In addition, the culture period may be a period during which each cell mass or cell arranged in the template internal space is integrated and a target three-dimensional cell structure is formed. Not limited, but usually 4 hours or more, 8 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more , 7 days or more, or 14 days or more, and less than 28 days, 25 days or less, 21 days or less, or 18 days or less. It is preferably 7 days or more and 21 days or less, and more preferably 14 days or more and 21 days or less. _{The CO 2} concentration at the time of culturing may be 4% or more, 4.5% or more, and 10% or less, or 5.5% or less, but is preferably 5%. In order to maintain good culture conditions, the medium can be changed at an appropriate frequency. Since the frequency of medium replacement varies depending on the cell type to be cultured, the type of medium, and the volume and shape of the mold internal space, it may be appropriately determined in consideration of these conditions. For example, it may be performed once every 5 days or more, once every 4 days or more, once every 3 days or more, once every 2 days or more, and once a day or more.

The flow state of the medium during culture does not matter. It may be a static culture or a fluid culture, but it is preferably a fluid culture.

"Standing culture" means culturing the medium in a static state in a culture tank. "Fluid culture" refers to culturing under conditions in which the medium is allowed to flow. Since the pores in the mold wall have liquid permeability, the medium can be naturally flowed in and out through the pores even in static culture, but the flow of the medium actively allows the medium to flow in and out of the mold internal space. Flow culture is preferable because it can be carried out easily. Examples of the flow culture method include a swirl culture method, a swing culture method, or a combination thereof.

"Swirl culture method" (including shaking culture method) refers to a method of culturing under conditions in which the medium flows in the culture tank due to stress (centrifugal force, centripetal force) due to the swirling flow. Specifically, the culture tank is swirled so as to draw a closed orbit such as a circle, an ellipse, a flat circle, or a flat ellipse along a horizontal plane, or the culture tank is left to stand and stirred with a stirring rod or agitator. A stirrer such as a wing can be used to swirl the medium in the tank to allow the medium to flow. The swirling speed in the swirling culture method is not particularly limited, but the lower limit may be 1 rpm or more, 10 rpm or more, 50 rpm or more, 60 rpm or more, 70 rpm or more, 80 rpm or more, 85 rpm or more, or 90 rpm or more. On the other hand, the upper limit can be 200 rpm or less, 150 rpm or less, 120 rpm or less, 115 rpm or less, 110 rpm or less, 105 rpm or less, 100 rpm or less, 95 rpm or less, or 90 rpm or less. The swirling width during swirling culture is not particularly limited, but the lower limit can be, for example, 1 mm or more, 10 mm or more, 20 mm or more, or 25 mm or more. On the other hand, the upper limit can be, for example, 200 mm or less, 100 mm or less, 50 mm or less, 30 mm or less, or 25 mm or less.

The "rocking culture method" refers to a method of culturing under conditions in which a rocking flow is applied to the medium by a linear reciprocating motion such as rocking stirring. Specifically, the culture tank is swung in a plane substantially perpendicular to the horizontal plane. The rocking speed is not particularly limited, but for example, when one round trip is once, the lower limit is 2 times, 4 times, 6 times, 8 times, or 10 times per minute, while the upper limit is 15 times per minute. It may swing 20 times, 25 times, or 50 times. When rocking, it is preferable to give the culture vessel a slight angle with respect to the vertical plane, that is, an induction angle. The swing angle is not particularly limited, but for example, the lower limit is 0.1 °, 2 °, 4 °, 6 °, or 8 °, while the upper limit is 10 °, 12 °, 15 °, 18 °, or 20 °. can do.
Furthermore, it is also possible to incubate while stirring by a motion that combines the above-mentioned turning and rocking.

(4) Separation Step The “separation step” is a step of separating the three-dimensional cell structure obtained after the culture step and the template. This process is a selective process. For example, when the template is composed of a material that can be fused with living tissue, for example, a polysaccharide polymer or a gelled protein, the three-dimensional cell structure after the culture step does not necessarily separate from the template and is in a state of being integrated with the template. It can also be used in. In this case, this step is unnecessary. On the other hand, when the template is an inanimate material such as metal or plastic, it is desirable to separate the template by this step after the culturing step and at least before using the produced three-dimensional cell structure.

The method for separating the three-dimensional cell structure from the template is not particularly limited. If the mold has an internal space, a three-dimensional cell structure, and an inner wall portion, a physical and / or chemical force may be applied between the inner wall portion and the three-dimensional cell structure, and both may be peeled off. Physical forces include, for example, shear stress, tensile force, pressure (including water pressure and air pressure), and temperature. Chemical forces include, for example, enzyme treatments such as weak proteolytic enzymes. When a physical force or a chemical external force is excessively applied to a cell, cell death can be induced, so the external force should be weak. When this step is performed, by making the cell adhesion surface such as the inner wall surface of the mold and the surface of the inner wall a non-cell adhesion material, the target three-dimensional cell structure is formed in the inner space of the mold, but is filled. Since the cell population does not adhere to the inner space wall surface of the mold or the surface of the inner wall portion, this step can be easily achieved with almost no external force applied.

After this step, the cell culture taken out from the template becomes the target three-dimensional cell structure obtained by the production method of the present invention.

1-4. Effect According to the method for producing a three-dimensional cell structure of the present invention, a three-dimensional cell structure having high cell density, shape uniformity and physical strength can be easily and stably produced in any shape. Can be done. As a result, the yield is improved and mass production of the three-dimensional cell structure becomes possible.

2. Three-dimensional cell structure 2-1. Overview A second aspect of the present invention is a three-dimensional cell structure. The three-dimensional cell structure of the present invention can be obtained by using the method for producing a three-dimensional cell structure of the first aspect. The three-dimensional cell structure obtained by the conventional method has a low cell density, tends to have a non-uniform shape, and has insufficient strength. However, according to the production method of the first aspect, the cells can be packed at high density by arranging the cell mass having a predetermined average diameter in the space inside the template, and then the cells are cultured together with the template. By doing so, it is possible to obtain an unprecedented three-dimensional cell structure having high cell density, shape uniformity, and physical strength.

2-2. Structure The structure of the three-dimensional cell structure of the present invention has a shape homologous to the shape of the internal space in the template according to the method for producing the three-dimensional cell structure of the first aspect. Therefore, it is possible to have an arbitrary shape depending on the mold. Specific examples of the shape of the three-dimensional cell structure of the present invention include a tubular structure that imitates a blood vessel described in Examples, and a structure having a complicated shape such as an auricle.

The cells constituting the structure are the cell population arranged in the template in the cell population arrangement step and the cells obtained by the culture step of culturing in the medium. Therefore, the type of cells constituting the structure depends on the cell mass and the type of cells contained in the cell population arranged at the time of production. In addition, individual cell masses and cells contained in a cell population are bound to each other by cell adhesion in the culture step to form an integrated cell aggregate.

The three-dimensional cell structure of the present invention has a significantly higher cell density than the conventional three-dimensional cell structure, and therefore has features of high shape uniformity and high physical strength.

2-3. Applications The three-dimensional cell structure of the present invention can be obtained by the production method of the first aspect and can be used as a member for transplantation in regenerative medicine. That is, according to the three-dimensional cell structure of the present invention, a three-dimensional cell structure used for a member for transplantation is provided. Further, according to the three-dimensional cell structure of the present invention, the use of the three-dimensional cell structure for manufacturing a member for transplantation is provided. Further, according to the three-dimensional cell structure of the present invention, a method of transplanting a three-dimensional cell structure into a patient or subject, including a step of administering a therapeutically effective amount of the three-dimensional cell structure to the patient or subject, and a patient. Alternatively, a method of treating the subject's disease is provided.

Examples of the transplanting member include International Publication WO2016 / 068292, and Anna D.A. Dikina, et al. , Biomaterials, 2015, 52: 452-462., Etc., known transplanting members suitable for artificial angioplasty and cartilage regeneration, and the three-dimensional cell structure of the present invention can also be used for such applications. it can.

The "patient or subject" in the present specification is typically a human, but may be another animal. Other animals include, for example, rodents such as mice, rats, hamsters, guinea pigs, snails, livestock or pets such as dogs, cats, rabbits, cows, horses, pigs, sheep, goats, ferrets, and humans. Examples include, but are not limited to, primates such as crab monkeys, red-tailed monkeys, common marmosets, Japanese monkeys, gorillas, and chimpanzees.

2-4. Effect Since the three-dimensional cell structure of the present invention has high cell density, shape uniformity and physical strength, it can be used as a member for transplantation in regenerative medicine.

A specific example of the method for producing a three-dimensional cell structure of the present invention is shown below. However, the production method of the present invention is not limited to the following methods.

<Method>
1. 1. Comparative Example 1
(1) Collection of fat Fat was collected from donor A who gave informed consent by the following method. A pre-specified collection site (abdomen, thigh, etc.) is incised by about 10 mm, and a cannula (φ2.5 / 260 mm: TP-203, MEDIKAN) and a disposable syringe (TP-101, MEDIKAN) are used to communicate. A solution (a mixture of 1 L of Japanese Pharmacopoeia physiological saline and 1 L of Japanese Pharmacopoeia adrenaline injection: 1 mg / 1 mL of Bosmin) was injected into the subcutaneous tissue for treatment. After the procedure, fat was aseptically sucked and collected using a cannula (φ4 / 260 mm: TP-201, φ3 / 260 mm: TP-202, MEDIKAN) connected to a disposable syringe (described above) (Lipokit, MEDICAN). .. Then, the fat was centrifuged under the condition of 700 × g for 5 minutes, and the supernatant was removed as a waste liquid.

(2) Enzyme treatment of fat and recovery of fat MSC The fat obtained in (1) above was immersed in a Hanks balanced salt solution (containing Ca / Mg) containing the same amount of 0.01% collagenase, and at 37 ° C. The fat was enzyme treated by stirring for 30 minutes at 200 rpm. The suspension of cells in which the cell adhesion was decomposed and dispersed after the treatment was centrifuged for 10 minutes under the condition of 800 × g, and the supernatant was removed.

Subsequently, αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing deactivated fetal bovine serum (FBS) having a final concentration of 10% was added and suspended, and then centrifuged under the condition of 800 × g for 5 minutes. , The supernatant was removed. The obtained precipitate was suspended in αMEM containing 10% FBS to obtain a cell suspension. This operation was repeated multiple times.

In order to separate and recover fat-derived MSCs (fat MSCs) from the obtained cell suspension, the ratio of cells positive for the surface antigen CD90 was analyzed using a flow cytometer with respect to the cell suspension. CD90 is a representative positive marker for MSCs. The surface antigen analysis was carried out using FACS canto of Becton Dickinson (BD), the number of analyzed cells: 10,000 cells, and the flow velocity setting: Medium. In this measurement, FITC Mouse IgG1, κ Isotype Control (BD / model number: 550616) was used as an antibody for isotype control. In addition, FITC Mouse Anti-Human CD90 (BD / model number: 555595) was used as an antibody against the CD90 antigen. As a result of the analysis, it was confirmed that the fat MSC could be separated from the cell suspension.

(3) Culturing of fat MSC A part of the cell population containing the fat MSC obtained in (2) above was seeded in ^{a culture vessel CellSTACK (registered trademark) (Corning Inc.) at a density of 4500 cells / cm 2.} The cells were adherently cultured in αMEM containing 10% deactivated FBS at the final concentration until they became subconfluent. These adherently cultured cells are referred to as "0th passage cell population". Subculture by exfoliating and collecting the 0th passage cell population from the culture vessel using TrypLE Select (Thermo Fisher Scientific) and seeding 1/5 amount of the cell population in CellSTACK® (described above). Was done. This first subcultured cell population is referred to as a "first subculture cell population". When the subconfluent was reached, the cell population of the first passage was exfoliated using TrypLE Select (described above), diluted with αMEM containing 10% deactivated FBS at the final concentration, and recovered by centrifugation. .. The collected cell population was suspended by adding a cryopreservation solution Bunbunker (registered trademark) (GC Lymphotech), and then cryopreserved under a -80 ° C deep freezer. Then, it was thawed and seeded in CellSTACK® (described above) at a density of ^{4500 cells / cm 2, and subcultured until it became subconfluent.} The cell population subcultured at this time is referred to as a "second passage cell population".

(4) Surface antigen analysis Each surface antigen (CD90 positive rate, CD73 positive rate, CD105 positive rate, CD45 positive rate and negative rate) was measured for the cell population of the second passage using a flow cytometer. did. Surface antigen analysis was performed using FACS canto (described above) with the number of cells analyzed: 10,000 cells and the flow velocity setting: Medium. In this measurement, FITC mouse IgG1, κ Mouse Control (described above) or PE mouse IgG1, κ Mouse Control (manufactured by BD / model number: 550617) was used as an antibody for isotype control. Further, PE anti-human CD90 (Th1) Antibody (manufactured by BioLegend / model number: 328110) was used as an antibody against the CD90 antigen, and PE Mouse Anti-Human CD73 (manufactured by BD / model number: 550257) was used as an antibody against the CD73 antigen. Anti-CD105 / FITC (manufactured by Ancell / model number: 326-040) was used as an antibody against the antigen, and FITC Mouse Anti-Human CD45 (manufactured by BD / model number: 555482) was used as an antibody against the CD45 antigen.

As a result of surface antigen analysis, in the cell population of the second passage, the positive rate of CD90, CD73 and CD105 was 80% or more, and the positive rate of CD45 was less than 5% (negative rate was 95% or more). .. From the above results, it was confirmed that the cell population of the second passage was a cell population containing adipose MSC.

(5) Preparation of cell mass A cell mass was prepared using the cell population of the second passage. First, αMEM containing deactivated FBS having a final concentration of 10% was used as a cell culture medium to prepare a cell suspension containing a second-generation cell population. Next, the cell suspension was seeded on a Prime Surface® plate 96U at 6.0 × 10 ^{4 cells per well.} Subsequently, the seeded plate was statically cultured at 37 ° C. for 24 hours under _{5% CO 2.} After culturing, the formation of cell clusters was confirmed using a microscope (manufactured by OLYMPUS). For the average diameter of the cell mass, after photographing the cells of the microscopic image using the imaging software cellSense (manufactured by OLYMPUS), the diameters of 60 cell masses are measured from any three directions based on the obtained photographed image. , Calculated. The magnification of the eyepiece at the time of photographing the microscope image was 10 times, and the magnification of the objective lens was 4 times.

(6) Preparation of template A template used in the method for producing a three-dimensional cell structure was prepared. FIG. 2 shows a conceptual diagram of the prepared mold (0200). As shown in this figure, the mold is a cylindrical bottom (0201) made of brass with a diameter of 5 mm and a porous mesh made of polypropylene (manufactured by Sampler Tech Co., Ltd., catalog number: WEB17815, hole major axis: 275.12 μm, average wire diameter: It has a substantially cylindrical wall portion (0202) composed of 140.74 μm and an aperture ratio of 43.8%). Further, a cylindrical inner wall portion (0203) having a diameter of 3 mm made of stainless steel is provided in the central portion of the inner space of the mold. The prepared mold has a cylindrical bottom portion (0201), a substantially cylindrical wall portion (0202), and a hollow portion (0204) surrounded by a cylindrical inner wall portion (0203). The mold was fixed to a silicon base (not shown).

(7) Production of Three-Dimensional Cell Structure The cell suspension containing 60 cell masses obtained in (5) above is flowed into the cavity (0204) formed in the template (0200) to form the cell mass. Randomly placed in the cavity. Then, the arranged cell mass was immersed in a cell culture medium together with the template, and statically cultured at 37 ° C. for 2 weeks under _{5% CO 2.} As the cell culture medium, αMEM containing deactivated FBS having a final concentration of 10% was used, and the medium was exchanged every two days during the above culture period.

(8) Separation of three-dimensional cell structure from the template The three-dimensional cell structure formed in the cavity after culturing was separated from the template. For separation, after cutting and removing the porous mesh of the template wall, the stainless steel cylinder of the inner wall is pulled out from the three-dimensional cell structure using tweezers, and tubular three-dimensional cells composed only of cells derived from the cell population. Obtained a structure.

2. Example 1
(1) Fat collection The basic operation followed the method described in "(1) Fat collection" of Comparative Example 1. However, in this example, fat was collected from donor B, which was different from Comparative Example 1 in which informed consent was obtained.
(2) Preparation of fat MSC From the enzyme treatment of the collected fat and the recovery of the fat MSC to the surface antigen analysis, the methods described in (2) to (4) of Comparative Example 1 were followed.
As a result of surface antigen analysis, the positive rate of CD90, CD73 and CD105 of the second passage cell population was 80% or more, and the positive rate of CD45 was less than 5% (negative rate was 95% or more). It was. From this result, it was confirmed that the cell population of the second passage of this example was a cell population containing adipose MSC.
(3) Preparation of cell mass The basic operation followed the method described in "(5) Preparation of cell mass" of Comparative Example 1. However, in this example, the plate after seeding the cell suspension was statically cultured for 96 hours.
(4) Preparation of Mold The basic operation followed the method described in "(6) Preparation of mold" of Comparative Example 1.
(5) Production of three-dimensional cell structure The basic operation followed the method described in "(7) Production of three-dimensional cell structure" of Comparative Example 1. However, in this example, 109 cell clusters were randomly arranged in the cavity.
(6) Separation of 3D cell structure from template The basic operation followed the method described in "(8) Separation of 3D cell structure from template" of Comparative Example 1.

3. 3. Example 2
(1) Fat collection In the basic operation, fat was collected from the same donor A as in Comparative Example 1 according to the method described in "(1) Fat collection" of Comparative Example 1.
(2) Preparation of fat MSC From the enzyme treatment of the collected fat and the recovery of the fat MSC to the surface antigen analysis, the methods described in (2) to (4) of Comparative Example 1 were followed. In the surface antigen analysis, the positive rates of CD90, CD73 and CD105 in the second passage cell population were all 80% or more (specifically, CD90: 100%, CD73: 98%, CD105: 85). %). The positive rate of CD45 was less than 5% (negative rate was 95% or more) (specifically, the positive rate of CD45: 0% (negative rate: 100%)). From the above results, it was confirmed that the cell population of the second passage of this example was a cell population containing adipose MSC.
(3) Preparation of cell mass The basic operation followed the method described in "(5) Preparation of cell mass" of Comparative Example 1. However, in this example, the cell suspension was seeded so as to ^{have 2.0 × 10 3} cells per well of the plate after seeding the cell suspension.
(4) Preparation of Mold The basic operation followed the method described in "(6) Preparation of mold" of Comparative Example 1. However, in this example, the holes of the porous mesh constituting the wall portion of the mold had a major axis of 275.12 μm and an average wire diameter of 140.74 μm. The aperture ratio of the porous mesh in the mold wall was calculated to be 43.8%.
(5) Production of three-dimensional cell structure The basic operation followed the method described in "(7) Production of three-dimensional cell structure" of Comparative Example 1. However, in this example, 138 cell masses obtained in the above-mentioned "(3) Preparation of cell mass" and 124 cell masses obtained in "(5) Preparation of cell mass" of Comparative Example 1 are used. They were mixed and randomly placed in the cavity.
(6) Separation of 3D cell structure from template The basic operation followed the method described in "(8) Separation of 3D cell structure from template" of Comparative Example 1.

<Result>
The results of Comparative Example 1, Example 1 and Example 2 are shown in Table 1 below.

In the table, in the "crack of cell culture", ○ indicates the case where the crack was confirmed, and × indicates the case where the crack could not be confirmed. Further, in the "ease of separation between the template and the three-dimensional cell structure", ◯ indicates a case where the separation was easy, and × indicates a case where the separation was difficult. Further, in the "acquisition of the target three-dimensional cell structure", ◯ indicates the case where the acquisition was possible, and × indicates the case where the acquisition was not possible. Then, in "homogeneity of the three-dimensional cell structure", ◯ indicates a case where it was uniform, and × indicates a case where it was non-uniform.

As shown in Table 1, the ratio (desired diameter cell mass content) of cell clusters having a desired diameter (average diameter of 50 μm or more and 750 μm or less) in the cell population in Example 1 was 100%. The desired diameter cell mass content in Example 2 was 53%. Furthermore, the desired diameter cell mass content in Comparative Example 1 was 0%.

As shown in Table 1, in Examples 1 and 2, the cell population containing the cell mass could be filled up to 60% or more of the volume of the template internal space (that is, the cell mass filling rate was 60% or more). However, in Comparative Example 1, the cell mass could not be filled at such a high density (that is, the cell mass filling rate was less than 60%).

Further, as shown in Tables 1, 3 and 4, in Examples 1 and 2, the cell clusters were uniformly fused in a high-density state in the culture step, and there were no visible cracks and a smooth surface was obtained. Formed as a cell culture with. On the other hand, as shown in Table 1 and FIG. 5, in Comparative Example 1, as a result of non-uniform fusion of cell masses in the culture step, a cell culture having visible cracks and a rough surface was formed. It was.

Further, as shown in Tables 1, 6 and 7, in Examples 1 and 2, the three-dimensional cell structure is taken out from the cavity formed by the mold internal space and the inner wall portion without applying a particularly large force. Was made. In particular, when the three-dimensional cell structure was removed from the stainless steel cylinder (the inner wall of the mold internal space), the three-dimensional cell structure could be grasped using tweezers, so that the three-dimensional cell structure was removed from the stainless steel cylinder. It could be easily separated. On the other hand, as shown in Table 1 and FIG. 8, in Comparative Example 1, although an attempt was made to remove the three-dimensional cell structure from the stainless steel cylinder, a large number of cracks were found on the surface of the three-dimensional cell structure. Due to the significant lack of physical strength, it was not possible to separate the 3D cell structure from the stainless steel cylinder while maintaining its original shape.

In addition, as shown in Tables 1 and 7, in Examples 1 and 2, the three-dimensional cell structure maintained its original shape even after separation from the template, and a desired tubular three-dimensional cell structure was obtained. We were able to. On the other hand, in Comparative Example 1, the three-dimensional cell structure was extremely brittle, and it was not possible to obtain a three-dimensional cell structure having a desired shape.

From the above, it was possible to efficiently produce a three-dimensional cell structure having high physical strength and a uniform shape by the production method of the present invention. Further, the three-dimensional cell structure obtained by the above-mentioned production method had high physical strength and a uniform shape.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (14)

1. It is a method for manufacturing a three-dimensional cell structure.
   It comprises a cell population placement step of arranging a cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less in the inner space of a template, and a culture step of culturing the cell population together with the template in a medium.
   The production method, wherein the wall portion of the mold has a porous structure through which liquid can pass.
2. The production method according to claim 1, wherein the cell population contains 50% or more of the cell mass.
3. The manufacturing method according to claim 1 or 2, wherein the internal space of the mold is cylindrical.
4. Claims 1 to 3 in which the ratio of the major axis (μm) and / or the maximum diagonal line (μm) of the pores in the porous structure to the average diameter (μm) of the cell mass is greater than 1.0 and less than or equal to 3.0. The manufacturing method according to any one of the above.
5. The production method according to any one of claims 1 to 4, wherein the pore opening ratio in the porous structure is 40% or more and 90% or less.
6. The production method according to any one of claims 1 to 5, wherein in the cell population arrangement step, the cell population containing the cell mass is filled to 60% or more and 95% or less of the volume of the mold internal space.
7. The production method according to any one of claims 1 to 6, wherein in the cell population placement step, the cell clusters are laminated without applying artificial pressure to the template.
8. The production method according to any one of claims 1 to 7, wherein the culture period in the culture step is 4 hours or more and less than 28 days.
9. The production method according to any one of claims 1 to 8, further comprising a separation step of separating the three-dimensional cell structure obtained after the culture step and the template.
10. The production method according to claim 9, wherein the cell contact surface of the template is made of a non-cell adhesive material.
11. The production method according to any one of claims 1 to 10, further comprising a cell population preparation step of preparing a cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less prior to the cell population arrangement step.
12. The production method according to any one of claims 1 to 11, wherein at least one cell constituting the cell mass is selected from the group consisting of mesenchymal stem cells, fibroblasts, endothelial cells and chondrocytes.
13. The production method according to claim 12, wherein the mesenchymal stem cells are derived from bone marrow, fat, fetal appendages or dental pulp.
14. It is a three-dimensional cell structure
    A cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less is placed in the inner space of the template, and then the cell population is cultured in a medium together with the template. The three-dimensional cell structure having a liquid-possible porous structure.

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